The neurotrophins are a family of structurally and functionally related proteins, including Nerve Growth Factor (NGF), Brain-Derived Neurotrophic Factor (BDNF), Neurotrophin-3 (NT-3), Neurotrophin-4/5 (NT-4/5) and Neurotrophin-6 (NT-6). These proteins promote the survival and differentiation of diverse neuronal populations in both the peripheral and central nervous systems and are involved in the pathogenesis of diverse neurological disorders (Hefti, J. Neurosci. 6:2155-2162 (1986); Hefti and Weiner, Annals of Neurology 20:275-281 (1986); Levi-Montalcini, EMBO J. 6:1145-1154 (1987); Barde, Neuron 2:1525-1534 (1989); Leibrock et al., Nature 341:149-152 (1989); Maisonpierre et al., Science 247:1446-1451 (1990); Rosenthal et al., Neuron 4:767-773 (1990); Hohn et al., Nature 344:339-341 (1990); Gotz et al., Nature 372:266-269 (1994); Maness et al., Neurosci. Biobehav. Rev. 18:143-159 (1994); Dechant et al., Nature Neurosci. 5:1131-1136 (2002)). This broad spectrum of biological activities exerted by the neurotrophins results from their ability to bind and activate two structurally unrelated receptor types, the p75 neurotrophin receptor (p75NTR) and the three members of the Trk receptor family of tyrosine kinases (Kaplan et al., Curr. Opin. Cell Biol. 9:213-221 (1997); Friedman et al., Exp. Cell Res. 253:131-142 (1999); Patapoutian et al., Curr. Opin. Neurobiol. 11:272-280 (2001)).
While NGF was initially studied for its essential role in neuronal growth and survival, recent reports indicate that this neurotrophin may also play a role in inflammation and disorders of the respiratory, the genitourinary and the gastrointestinal systems. For example, in the gastrointestinal tract, neurotrophins and neurotrophic factors regulate neuropeptide expression, interact with immunoregulatory cells and epithelial cells, and regulate motility during inflammation (Reinshagen, M. et al., Curr. Opin. Investig. Drugs. 2002; 3(4): 565-568). NGF has been shown to play a role in bladder overactivity (Lamb, K. et al., J. Pain. 2004; 5(3): 150-156), bladder outlet obstruction (Kim, J. C. et al., BJU Int. 2004; 94(6): 915-918), pancreatic cancer (Shi, X. et al., Pancreatology. 2001; 1(5):517-524), and intestinal inflammation (Lin, A. et al., Exp. Neurol. 2005; 191(2):337-43).
NGF is synthesized as a larger precursor form (referred to herein as “proNGF,” also known as “preproNGF” or “pro-peptide NGF”) which is then processed by proteolytic cleavages to produce the mature neurotrophic factor. This prepro region is located at the amino terminus of the precursor molecule and is needed for proper folding and secretion of the NGF protein. The mature form of NGF has arginine residues at its carboxy termini which requires that a leucine residue be inserted between the naturally occurring arginine and the hydrophilic spacer. The primary structure of proNGF has been deduced from the nucleotide sequence of the mouse NGF cDNA (Scott et al. Nature 302:538 (1983); Ullrich et al. Nature 303:821 (1983)).
The common neurotrophin receptor p75NTR is a transmembrane glycoprotein structurally related to the tumor necrosis factor and CD-40 receptors (Meakin and Shooter, Trends Neurosci. 15:323-331 (1992), Rydén and Ibáñez, J. Biol. Chem. 271:5623-5627 (1996)). As all neurotrophins bind to p75NTR with similar affinities (Rodrigues-Tébar et al., Neuron 4:487-492 (1990); Hallbook et al., Neuron 6:845-858 (1991); Rodrigues-Tébar et al., EMBO J. 11:917-922 (1992); Ibáñez, Trends Biotech. 13:217-227 (1995)), neurotrophin specificity is conventionally thought to be conferred by the binding selectivity for Trk receptors which are differentially expressed in different neuronal populations (Ibáñez, Trends Biotech. 13:217-227 (1995)). However, accumulated experimental data on neurotrophin activity reveal important functional aspects of p75NTR (Heldin et al., J. Biol. Chem. 264:8905-8912 (1989); Jing et al., Neuron 9:1067-1079 (1992); Herrmann et al., Mol. Biol. 4:1205-1216 (1993); Barker and Shooter, Neuron 13:203-215 (1994); Dobrowsky et al., Science 265:1596-1599 (1994), Matsumoto et al., Cancer Res. 55:1798-1806 (1995); Marchetti et al., Cancer Res. 56:2856-2863 (1996); Washiyama et al., Amer. J. Path. 148:929-940 (1996)). The common neurotrophin receptor enhances functions and increases binding specificity of Trk receptors (Barker and Shooter, Neuron 13:203-215 (1994); Mahadeo et al., J. Biol. Chem. 269:6884-6891 (1994); Chao and Hempstead, Trends Neurosci. 18:321-326 (1995); Rydén and Ibáñez, J. Biol. Chem. 271:5623-5627 (1996)). In addition, p75NTR possesses unique, neurotrophin dependent, Trk-independent signaling properties which involve ceramide production through activation of the sphingomyelin cycle (Dobrowsky et al., Science 265:1596-1599 (1994)), apoptosis (cell death) (Cassacia-Bonnefil et al., Nature 383:716-719 (1996)), and activation of the transcription factor NFKB (Carter et al., Science 272:542-545 (1996)).
Moreover, while initially studied primarily in neurons, p75NTR has also been found to play critical roles in vascular biology (von Schack et al., Nat. Neurosci. 4:977-978, 2001; Wan et al., Am. J. Pathol. 157:1247-1258, 2001), glial biology (Bentley et al., J. Neurosci. 20:7706-7715, 2000; Syroid et al., J. Neurosci. 20:5741-5747, 2000), the immune system (Tokuoka et al., Br. J. Pharmacol. 134:1580-1586, 2001), and tumor biology (Sakamoti et al., Oncol. Rep. 8:973-980, 2001; Descamps et al., J. Biol. Chem. 276:17864017870, 2001). For example, p75NTR has been demonstrated to participate in human melanoma progression (Herrmann et al., Mol. Biol. 4:1205-1216 (1993); Marchetti et al., Cancer Res. 56:2856-2863 (1996)). Furthermore, NGF and NT-3 increase the production of heparin by 70 W melanoma cells, which is associated with their metastatic potential (Marchetti et al., Cancer Res. 56:2856-2863 (1996)).
Unlike p75NTR, the Trk receptors (TrkA, TrkB and TrkC) exhibit selectivity for specific neurotrophins. (Kaplan et al., Science 252:554-558 (1991); Klein et al., Cell 65:189-197 (1991); Klein et al., Neuron 8:947-956 (1992); Soppet et al., Cell 65:895-903 (1991); Squinto et al., Cell 65:885-893 (1991); Berkemeier et al., Neuron 7:857-866 (1991); Escandon et al., Neurosci. Res. 34:601-613 (1993); Lamballe et al., Cell 66:967-970 (1991)). For example, TrkA primarily binds NGF (Kaplan et al., 1991; Klein et al., 1991) and has been reported to bind NT-3 (J. Biol. Chem. 271(10):5623-7, 1996); TrkB binds BDNF and NT-4/5 (Soppet et al., 1991; Squinto et al., 1991; Berkemeier et al., 1991; Escandon et al., 1993; Lamballe et al., 1991; Klein et al., 1992; Vale and Shooter, Methods Enzymol. 109:21-39 (1985); Barbacid, Oncogene 8:2033-2042 (1993)); and TrkC exclusively binds NT-3 (Lamballe et al., 1991; Vale and Shooter, 1985). This is particularly evident when the Trk receptors are coexpressed with the common neurotrophin receptor p75NTR. (For review see Meakin and Shooter, 1992; Barbacid, 1993; Chao, 1994; Bradshaw et al., 1994; Ibáñez, 1995).
Biochemical experiments indicate that neurotrophin receptors form at least three different types of complexes: homodimers of Trk receptors, homomeric p75NTR receptors and mixed complexes of both Trk and p75NTR. These complexes may coexist in cells and may be linked through biochemical equilibria. Functionally, their signaling has been shown to be independent, synergistic or antagonistic. The response of a cell to neurotrophins is thus determined by the quantitative and qualitative composition of its receptor complement in combination with biochemical equilibria between pools of active and inactive receptors (Dechant, Cell Tissue Res. 305:229-238, (2001)), as well as other cellular and biochemical components downstream of the neurotrophin receptors, e.g., the availability of proteins, lipids and inorganic molecules involved in signal transduction.
Due to the implication of NGF, and its precursor proNGF, binding to homomeric and heteromeric neurotrophin receptor complexes in various disease states, especially pain, inflammation, neurological disorders and disorders of the respiratory, genitourinary and gastrointestinal systems, a need exists for pharmaceutical agents and methods of use thereof for modulating the interactions of NGF with the common neurotrophin receptor p75NTR, and the Trk receptor TrkA.